Up to 2-5% of all prenatal ultrasounds identify hydronephrosis. Ureteropelvic junction obstruction (UPJO) typically manifests with hydronephrosis, but not all patients with hydronephrosis have clinically significant obstruction, and many spontaneously resolve. Patients typically undergo extensive serial invasive imaging to identify which children require surgical intervention, as renal obstruction can lead to abnormal renal maturation or loss of kidney function. None of these current imaging methods are predictive. Without doubt, development of a stratifying biomarker capable of identifying children with hydronephrosis who are at- risk for renal damage is the most pressing clinical need, since early diagnosis and surgical intervention can prevent progressive renal damage, and better stratification would reduce invasive imaging. Our initial funding period has produced a ?best? panel of 34 non-invasive urinary biomarkers that have the ability to identify children with hydronephrosis who have clinical parameters consistent with renal dysfunction. Moreover, these markers improved after UPJO correction demonstrating their significant potential to be used serially during longitudinal observation of hydronephrosis. Functional assessment of the novel putative biomarkers as a non-invasive assessment of stress-related damage at the sub-cellular level demonstrated a simultaneous regulation of Ca2+ signaling and production of nitrogen oxide (NO) with previously-observed response to reactive oxygen species (ROS) during renal damage, as well as modulators of matrix metalloproteinases related to extracellular matrix remodeling (ECM). From this data, we hypothesize that these biomarkers will identify patients with hydronephrosis at-risk for renal damage and will function as early markers of ongoing renal dysfunction prior to clinical radiographic changes. We predict that these non-invasive biomarkers in combination with ultrasound could become the new paradigm for indicating when additional imaging or surgical intervention is needed. We will challenge this hypothesis with the following specific aims: 1): Validate the identified candidate biomarkers in a new study cohort of UPJO patients to distinguish at-risk patients with hydronephrosis. 2) Determine and validate which candidate markers can predict renal dysfunction in a longitudinal observation cohort. This proposal will rigorously validate urinary biomarkers that have the potential to substantially improve the management of a very large population of children with hydronephrosis. These novel studies will be the first state-of-the-art validation of the candidate markers as a UPJO diagnostic tool. These highly translational experiments are of direct relevance to human health, as the findings may develop a new clinical paradigm that has the ultimate potential of decreasing invasive imaging and improve surgical decision making.